Sophocarpine attenuates toll-like receptor 4 in steatotic hepatocytes to suppress pro-inflammatory cytokines synthesis.

BACKGROUND AND AIM: Sophocarpine, a tetracyclic quinolizidine alkaloid derived from Sophora alopecuroides L., has been documented that it can suppress pro-inflammatory cytokines synthesis in alleviating nonalcoholic steatohepatitis (NASH) in vivo. Toll-like receptor 4 (TLR4) is a pattern recognition receptor whose activation results in the production of several pro-inflammatory cytokines. It has been reported that TLR4 is upregulated in nonalcoholic fatty liver disease and plays an important role in the pathogenesis of NASH. This study aimed to examine the changes of TLR4 and its signaling pathways in sophocarpine's anti-inflammatory process on experimental NASH in vitro. METHODS: Primary hepatocytes were isolated, and oleic acid-induced steatosis model was established. Cell Counting Kit-8 assay was used to detect the number of metabolically active mitochondria and viable cells. Immunocytochemistry analysis was applied to evaluating pro-inflammatory cytokines synthesis. Total RNA and protein were extracted for real-time polymerase chain reaction and Western blot detection. RESULTS: Enhanced expression of TLR4 was observed in oleic acid-induced steatotic hepatocytes. Sophocarpine suppressed pro-inflammatory cytokines synthesis and reduced the expression of TLR4 in steatotic hepatocytes. Expression of TLR4 and pro-inflammatory cytokines recovered after sophocarpine was removed. Moreover, sophocarpine restrained the activation of nuclear factor-kappaB (NF-κB), c-Jun-N-terminal kinase (JNK), and Extracellular regulated protein kinases (ERK) signaling pathways in the anti-inflammatory process. CONCLUSION: Sophocarpine could decrease the expression of TLR4 in steatotic hepatocytes and suppress pro-inflammatory cytokines synthesis. NF-κB, JNK, and ERK signaling pathways were important workable downstream pathways.

[Mechanism of sophocarpine in treating experimental colitis in mice].

To study the preventive effect of sophocarpine (Soc) on dextran sulfate sodium (DSS)-induced colitis in mice, in order to analyze the influence of Soc on toll like receptor 4 (TLR4)/mitogen-activated protein kinases (MAPKs) and janus tyrosine kinase 2 signal transducer and activator of transcription 3 (JAK2/STAT3) signal pathways in mice intestinal tissues. The mice was given 2.5% DSS for 6 days to induce the acute colitis model. The Soc-treated group was intraperitoneally injected with sophocarpine 30 mg · kg(-1) · d(-1) since the day before the experiment to the end. The disease activity index (DAI) was assessed everyday, and the colonic morphology and histological damage were observed with HE staining. The mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were detected by real-time RT-PCR. The changes in key protein kinase p38 mitogen-activated protein kinase (p38MAPK), c-Jun NH2-terminal protein kinase1/2 (JNK1/2), extracellular signal-regulated kinase1/2 (ERK1/2), JAK2, STAT3 in TLR4/MAPKs and JAK2/STAT3 signaling pathways were detected by western blot. The result showed that the model group showed statistical significance in body weight, DAI, colon length and histopathological changes compared with the normal group (P <0.05); however, the Soc-treated group showed significant improvements in the above indexes compared with the model group (P <0.05). TNF-α, IL-1ß and IL-6 in the model group was significantly higher than that in the normal group (P <0.05), but lowered in the Soc-treated group to varying degrees (P <0.05). In the normal group, the expressions of TLR4 and the phosphorylation of P38, JNK1/2, JAK2, STAT3 were at low levels; in the model group, the phosphorylation of P38, JNK1/2, JAK2, STAT3 increased; the Soc-treated group showed a decrease in TLR4 expression compared with the model group, with notable declines in the phosphorylation of TLR4, P38, JNK1/2, JAK2, STAT3. These findings indicate that Soc can inhibit TLR4/MAPKs, K2/STAT3 signaling pathway activation, reduce the expression of proinflammatory cytokines TNF-α, IL-1ß and IL-6 and relieve inflammatory reactions, so as to effectively prevent experimental colitis.

Perturbation of MicroRNA-370/Lin-28 homolog A/nuclear factor kappa B regulatory circuit contributes to the development of hepatocellular carcinoma.

UNLABELLED: MicroRNA 370 (miR-370) is located within the DLK1/DIO3 imprinting region on human chromosome 14, which has been identified as a cancer-associated genomic region. However, the role of miR-370 in malignances remains controversial. Here, we report that miR-370 was repressed in human hepatocellular carcinoma (HCC) tissues and hepatoma cell lines. Using gain-of-function and loss-of-function experiments, we demonstrated that miR-370 inhibited the malignant phenotype of HCC cells in vitro. Overexpression of miR-370 inhibited growth and metastasis of HCC cells in vivo. Moreover, the RNA-binding protein, LIN28A, was identified as a direct functional target of miR-370, which, in turn, blocked the biogenesis of miR-370 by binding to its precursor. LIN28A also mediated the suppressive effects of miR-370 on migration and invasion of HCC cells by post-transcriptionally regulating RelA/p65, which is an important effector of the canonical nuclear factor kappa B (NF-κB) pathway. Interleukin-6 (IL-6), a well-known NF-κB downstream inflammatory molecule, reduced miR-370 but increased LIN28A levels in HCC. Furthermore, miR-370 levels were inversely correlated with LIN28A and IL-6 messenger RNA (mRNA) levels, whereas LIN28A mRNA expression was positively correlated with IL-6 expression in human HCC samples. Interestingly, reduction of miR-370 expression was associated with the development of HCC in rats, as well as with aggressive tumor behavior and short survival in HCC patients. CONCLUSIONS: These data demonstrate the involvement of a novel regulatory circuit consisting of miR-370, LIN28A, RelA/p65 and IL-6 in HCC progression. Manipulating this feedback loop may have beneficial effect in HCC treatment.

Evaluation and clinical significance of the stomach age model for evaluating aging of the stomach-a multicenter study in China.

BACKGROUND: A higher prevalence of chronic atrophic gastritis (CAG) occurs in younger adults in Asia. We used Stomach Age to examine the different mechanisms of CAG between younger adults and elderly individuals, and established a simple model of cancer risk that can be applied to CAG surveillance. METHODS: Stomach Age was determined by FISH examination of telomere length in stomach biopsies. Δψm was also determined by flow cytometry. Sixty volunteers were used to confirm the linear relationship between telomere length and age while 120 subjects were used to build a mathematical model by a multivariate analysis. Overall, 146 subjects were used to evaluate the validity of the model, and 1,007 subjects were used to evaluate the relationship between prognosis and Δage (calculated from the mathematical model). ROC curves were used to evaluate the relationship between prognosis and Δage and to determine the cut-off point for Δage. RESULTS: We established that a tight linear relationship between the telomere length and the age. The telomere length was obvious different between patients with and without CAG even in the same age. Δψm decreased in individuals whose Stomach Age was greater than real age, especially in younger adults. A mathematical model of Stomach Age (real age + Δage) was successfully constructed which was easy to apply in clinical work. A higher Δage was correlated with a worse outcome. The criterion of Δage >3.11 should be considered as the cut-off to select the subgroup of patients who require endoscopic surveillance. CONCLUSION: Variation in Stomach Age between individuals of the same biological age was confirmed. Attention should be paid to those with a greater Stomach Age, especially in younger adults. The Δage in the Simple Model can be used as a criterion to select CAG patients for gastric cancer surveillance.

Concurrent hepatopathy in dogs with gallbladder mucocele: Prevalence, predictors, and impact on long-term outcome.

BACKGROUND: Information is limited regarding the prevalence and importance of hepatic histologic abnormalities in dogs with gallbladder mucocele (GBM). OBJECTIVES: To (a) report prevalence of hepatic histologic abnormalities in dogs with GBM (b) evaluate for association between hepatic abnormalities and outcome in dogs with GBM (c) evaluate whether neutrophil-to-lymphocyte ratio (NLR) differs in dogs with GBM with and without specific hepatic lesions. ANIMALS: Fifty-two dogs with grossly and histologically confirmed GBM. METHODS: Multicenter, retrospective study of dogs with GBM undergoing cholecystectomy with concurrent liver biopsy. Archived histological sections of gallbladder and liver evaluated by investigators blinded to data. Proportions of dogs with each histologic abnormality alive vs deceased at 1, 3, and 12 months post-cholecystectomy compared. Mann-Whitney U performed to determine if NLR differed in dogs with or without selected lesions. RESULTS: 51/52 (98%, 95% CI [89%, 99%]) dogs with GBM had at least 1 hepatic histologic abnormality. Hepatic fibrosis (37/51; 73%, 95% CI [59%, 83%]), biliary hyperplasia (29/52; 56%, 95% CI [42%, 68%]), and portal inflammation (25/52; 48%, 95% CI [35%, 61%]) were most common. The proportion of dogs alive vs dead differed based on the fibrosis score at 1, 3, and 12 (P ≤ .04) months post-cholecystectomy. Dogs with hepatic necrosis (P = .006) and cholangitis/cholangiohepatitis (P = .02) had higher NLRs compared to dogs without these lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: Histologic abnormalities of the liver are common in dogs with GBM. A higher portal fibrosis score might be associated with shortened long-term survival after cholecystectomy for dogs with GBM. An increase in NLR might predict hepatic necrosis and cholangitis/cholangiohepatitis in dogs with GBM.

Skeletal muscle alterations indicate poor prognosis in cirrhotic patients: a multicenter cohort study in China.

INTRODUCTION: We aimed to determine the diagnostic criteria of myosteatosis in a Chinese population and investigate the effect of skeletal muscle abnormalities on the outcomes of cirrhotic patients. METHODS: Totally 911 volunteers were recruited to determine the diagnostic criteria and impact factors of myosteatosis, and 480 cirrhotic patients were enrolled to verify the value of muscle alterations for prognosis prediction and establish new noninvasive prognostic strategies. RESULTS: Multivariate analysis showed age, sex, weight, waist circumference, and biceps circumference had a remarkable influence on the L3 skeletal muscle density (L3-SMD). Based on the cut-off of a mean - 1.28 × SD among adults aged < 60 years, the diagnostic criteria for myosteatosis was L3-SMD < 38.93 Hu in males and L3-SMD < 32.82 Hu in females. Myosteatosis rather than sarcopenia has a close correlation with portal hypertension. The concurrence of sarcopenia and myosteatosis not only is associated with poor liver function but also evidently reduced the overall and liver transplantation-free survival of cirrhotic patients (p < 0.001). According to the stepwise Cox regression hazard model analysis, we established nomograms including TBil, albumin, history of HE, ascites grade, sarcopenia, and myosteatosis for easily determining survival probabilities in cirrhotic patients. The AUC is 0.874 (95% CI 0.800-0.949) for 6-month survival, 0.831 (95% CI 0.764-0.898) for 1-year survival, and 0.813 (95% CI 0.756-0.871) for 2-year survival prediction, respectively. CONCLUSIONS: This study provides evidence of the significant correlation between skeletal muscle alterations and poor outcomes of cirrhosis, and establishes valid and convenient nomograms incorporating musculoskeletal disorders for the prognostic prediction of liver cirrhosis. Further large-scale prospective studies are necessary to verify the value of the nomograms.

Biocompatibility and Safety Assessment of Combined Topical Ozone and Antibiotics for Treatment of Infected Wounds.

Wound infections with antibiotic-resistant bacteria, particularly the Gram-negative strains, pose a substantial health risk for patients with limited treatment options. Recently topical administration of gaseous ozone and its combination with antibiotics through portable systems has been demonstrated to be a promising approach to eradicate commonly found Gram-negative strains of bacteria in wound infections. However, despite the significant impact of ozone in treating the growing number of antibiotic-resistant infections, uncontrolled and high concentrations of ozone can cause damage to the surrounding tissue. Hence, before such treatments could advance into clinical usage, it is paramount to identify appropriate levels of topical ozone that are effective in treating bacterial infections and safe for use in topical administration. To address this concern, we have conducted a series of in vivo studies to evaluate the efficacy and safety of a portable and wearable adjunct ozone and antibiotic wound therapy system. The concurrent ozone and antibiotics are applied through a wound interfaced gas permeable dressing coated with water-soluble nanofibers containing vancomycin and linezolid (traditionally used to treat Gram-positive infections) and connected to a portable ozone delivery system. The bactericidal properties of the combination therapy were evaluated on an ex vivo wound model infected with Pseudomonas aeruginosa, a common Gram-negative strain of bacteria found in many skin infections with high resistance to a wide range of currently available antibiotics. The results indicated that the optimized combination delivery of ozone (4 mg h-1) and topical antibiotic (200 µg cm-2) provided complete bacteria eradication after 6 h of treatment while having minimum cytotoxicity to human fibroblast cells. Furthermore, in vivo local and systemic toxicity studies (e.g., skin monitoring, skin histopathology, and blood analysis) on pig models showed no signs of adverse effects of ozone and antibiotic combination therapy even after 5 days of continuous administration. The confirmed efficacy and biosafety profile of the adjunct ozone and antibiotic therapy places it as a strong candidate for treating wound infection with antimicrobial-resistant bacteria and further pursuing human clinical trials.

Covert hepatic encephalopathy is associated with aggressive disease progression and poor survival in patients with cirrhosis.

OBJECTIVES: Covert hepatic encephalopathy (CHE) negatively affects the health-related quality of life and increases the risk of overt HE (OHE) in patients with liver cirrhosis. However, the impact of CHE on long-term patient outcomes remains controversial. This study aimed to explore the association between CHE and disease progression and survival among cirrhotic patients. METHODS: This was a single-center prospective study that enrolled 132 hospitalized patients with cirrhosis, with an average follow-up period of 45.02 ± 23.06 months. CHE was diagnosed using the validated Chinese standardized psychometric hepatic encephalopathy score. RESULTS: CHE was detected in 35.61% cirrhotic patients. During the follow-up, patients with CHE had a higher risk of developing OHE (log-rank 5.840, P = 0.016), exacerbation of ascites (log-rank 4.789, P = 0.029), and portal vein thrombosis (PVT) (log-rank 8.738, P = 0.003). Cox multivariate regression analyses revealed that CHE was independently associated with the occurrence of OHE, exacerbation of ascites, and PVT. Furthermore, patients with progression of cirrhosis were more likely to be diagnosed as CHE (log-rank 4.462, P = 0.035). At the end of the follow-up, patients with CHE had a lower survival rate compared to those without CHE (log-rank 8.151, P = 0.004). CHE diagnosis (hazard ratio 2.530, P = 0.008), together with elder age and higher Child-Pugh score, were risk factors for impaired survival in cirrhotic patients. CONCLUSION: CHE is associated with disease progression and poor survival in patients with cirrhosis, indicating that CHE may serve as an independent predictor of poor prognosis among these patients.

Recombinant adenovirus carrying the hepatocyte nuclear factor-1alpha gene inhibits hepatocellular carcinoma xenograft growth in mice.

UNLABELLED: Hepatocyte nuclear factor-1alpha (HNF1α) is one of the key transcription factors of the HNF family, which plays a critical role in hepatocyte differentiation. Substantial evidence has suggested that down-regulation of HNF1α may contribute to the development of hepatocellular carcinoma (HCC). Herein, human cancer cells and tumor-associated fibroblasts (TAFs) were isolated from human HCC tissues, respectively. A recombinant adenovirus carrying the HNF1α gene (AdHNF1α) was constructed to determine its effect on HCC in vitro and in vivo. Our results demonstrated that HCC cells and HCC tissues revealed reduced expression of HNF1α. Forced reexpression of HNF1α significantly suppressed the proliferation of HCC cells and TAFs and inhibited the clonogenic growth of hepatoma cells in vitro. In parallel, HNF1α overexpression reestablished the expression of certain liver-specific genes and microRNA 192 and 194 levels, with a resultant increase in p21 levels and induction of G(2)/M arrest. Additionally, AdHNF1α inhibited the expression of cluster of differentiation 133 and epithelial cell adhesion molecule and the signal pathways of the mammalian target of rapamycin and transforming growth factor beta/Smads. Furthermore, HNF1α abolished the tumorigenicity of hepatoma cells in vivo. Most interestingly, intratumoral injection of AdHNF1α significantly inhibited the growth of subcutaneous HCC xenografts in nude mice. Systemic delivery of AdHNF1α could eradicate the orthotopic liver HCC nodules in nonobese diabetic/severe combined immunodeficiency mice. CONCLUSION: These results suggest that the potent inhibitive effect of HNF1α on HCC is attained by inducing the differentiation of hepatoma cells into mature hepatocytes and G(2)/M arrest. HNF1α might represent a novel, promising therapeutic agent for human HCC treatment. Our findings also encourage the evaluation of differentiation therapy for tumors of organs other than liver using their corresponding differentiation-determining transcription factor.

Self-expanding metallic stent as a bridge to surgery versus emergency surgery for obstructive colorectal cancer: a meta-analysis.

BACKGROUND: The use of a colonic stent as a bridge to surgery aims to provide patients with elective one-stage surgical resection while reducing stoma creation and postoperative complications. This study used meta-analytic techniques to compare the outcomes of stent use as a bridge to surgery and emergency surgery in the management of obstructive colorectal cancer. METHODS: A literature search of Medline, Embase, Cochrane controlled trials registry, and the Chinese Biomedical Literature Database was performed on all studies comparing stent as a bridge to surgery and emergency surgery for obstructive colorectal cancer. A meta-analysis of the included studies was carried out to identify the differences in outcomes between the two procedures. RESULTS: Eight studies matched the criteria for inclusion and reported on the outcomes of 601 patients, of whom 232 (38.6%) underwent stent insertion and 369 (61.4%) underwent emergency surgery. Fewer patients in the stent group needed intensive care (risk ratio [RR], 0.42; 95% confidence interval [CI], 0.19-0.93; p = 0.03) and stoma creation (RR, 0.70; 95% CI, 0.50-0.99; p = 0.04). The primary anastomosis rate in the stent group was higher (RR, 1.62; 95% CI, 1.21-2.16; p = 0.001). Overall complications (RR, 0.42; 95% CI, 0.24-0.71; p = 0.001), including anastomotic leakage (RR, 0.31; 95% CI, 0.14-0.69; p = 0.004), were reduced by stent insertion. Stent placement before elective surgery did not adversely affect mortality and long-term survival. CONCLUSIONS: The use of a stent as a bridge to surgery for obstructive left-sided colorectal cancer could increase the chance of primary anastomosis and reduce the need for stoma creation and postprocedural complications. Stent insertion before subsequent surgery has no effect on perioperative mortality and long-term survival.

Sophocarpine alleviates non-alcoholic steatohepatitis in rats.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) is one entity in the spectrum of non-alcoholic fatty liver disease (NAFLD). The aim of this study was to explore the prevention and therapeutic effect of sophocarpine on experimental rat NASH. METHODS: Sophocarpine with the dosage of 20 mg/kg/day was injected into NASH rats. At the end of 12 weeks, all rats were killed to detect the degree of fatty degeneration, inflammation and fibrosis. RESULTS: Sophocarpine intervention (in the pro-treated and treated groups) resulted in a significant decrease of liver weight, liver index, serum transaminase and serum lipids. Messenger RNA expressions of leptin, interleukin (IL)-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1, procollagen-I and α-smooth muscle actin (SMA) and deposition of IL-6, TNF-α and TGF-ß1 in liver decreased, whereas the messenger RNA expression of adiponectin increased significantly compared with that in the model group. Moreover, histological improvement was also observed in the sophocarpine intervention group. In addition, there was no significant difference in any detected indicator between the pro-treated and treated group. CONCLUSIONS: Sophocarpine could decrease the level of serum transaminase, improve lipid metabolism, reduce synthesis of inflammatory cytokines TNF-α, TGF-ß1 and IL-6, activate protective adipocytokine adiponectin, and might be selected as a promising agent for the clinical prevention and therapy of NASH.

Inhibition of extracellular signal-regulated kinase 1 by adenovirus mediated small interfering RNA attenuates hepatic fibrosis in rats.

UNLABELLED: Extracellular signal-regulated kinase 1 (ERK1) is a critical part of the mitogen-activated protein kinase signal transduction pathway, which is involved in hepatic fibrosis. However, the effect of down-regulation of ERK1 on hepatic fibrosis has not been reported. Here, we induced hepatic fibrosis in rats with dimethylnitrosamine administration or bile duct ligation. An adenovirus carrying small interfering RNA targeting ERK1 (AdshERK1) was constructed to determine its effect on hepatic fibrosis, as evaluated by histological and immunohistochemical examination. Our results demonstrated that AdshERK1 significantly reduced the expression of ERK1 and suppressed proliferation and levels of fibrosis-related genes in hepatic stellate cells in vitro. More importantly, selective inhibition of ERK1 remarkably attenuated the deposition of the extracellular matrix in fibrotic liver in both fibrosis models. In addition, both hepatocytes and biliary epithelial cells were proven to exert the ability to generate the myofibroblasts depending on the insults of the liver, which were remarkably reduced by AdshERK1. Furthermore, up-regulation of ERK1 paralleled the increased expression of transforming growth factor beta1 (TGF-beta1), vimentin, snail, platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 4 (BMP4), and small mothers against decapentaplegic-1 (p-Smad1), and was in reverse correlation with E-cadherin in the fibrotic liver. Nevertheless, inhibition of ERK1 resulted in the increased level of E-cadherin in parallel with suppression of TGF-beta1, vimentin, snail, PDGF-BB, BMP4, and p-Smad1. Interestingly, AdshERK1 treatment promoted hepatocellular proliferation. CONCLUSION: Our study provides the first evidence for AdshERK1 suppression of hepatic fibrosis through the reversal of epithelial-mesenchymal transition of both hepatocytes and biliary epithelial cells without interference of hepatocellular proliferation. This suggests that ERK1 is implicated in hepatic fibrogenesis and selective inhibition of ERK1 by small interfering RNA may present a novel option for hepatic fibrosis treatment.

Adenovirus-mediated transfer of siRNA against PAI-1 mRNA ameliorates hepatic fibrosis in rats.

BACKGROUND/AIMS: Plasminogen activator inhibitor-1 (PAI-1) is a potential profibrotic molecule. The aim of this study was to evaluate the therapeutic effect of PAI-1 small interfering RNA (siRNA) on experimental hepatic fibrosis and investigate the intrinsic mechanisms. METHODS: Hepatic fibrosis in rats was induced by dimethylnitrosamine (DMN) administration or bile duct ligation (BDL). An adenovirus carrying PAI-1 shRNA (AdshPAI) was generated and administered via tail vein injection. The expression of PAI-1 was confirmed by real-time RT-PCR and immunohistochemistry. The effect of AdshPAI on fibrosis was evaluated by histological and immunohistochemical examination. RESULTS: We found that PAI-1 was downregulated after AdshPAI administration. Liver fibrosis was significantly improved after AdshPAI administration in both DMN and BDL models. AdshPAI treatment facilitated matrix degradation by correcting the levels of matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) through upregulation of MMP9, MMP13 and downregulation of TIMP-1. Moreover, AdshPAI treatment stimulated hepatocellular proliferation and inhibited cellular apoptosis. CONCLUSIONS: This study suggests that AdshPAI treatment has a protective effect on hepatocytes and ameliorates liver fibrogenesis. Inhibiting the upregulation of PAI-1 during liver fibrosis may be an antifibrotic pathway worth exploiting.

Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4alpha gene.

UNLABELLED: Previous studies have shown that hepatocyte nuclear factor-4alpha (HNF4alpha) is a central regulator of differentiated hepatocyte phenotype and forced expression of HNF4alpha could promote reversion of tumors toward a less invasive phenotype. However, the effect of HNF4alpha on cancer stem cells (CSCs) and the treatment of hepatocellular carcinoma (HCC) with HNF4alpha have not been reported. In this study, an adenovirus-mediated gene delivery system, which could efficiently transfer and express HNF4alpha, was generated to determine its effect on hepatoma cells (Hep3B and HepG2) in vitro and investigate the anti-tumor effect of HNF4alpha in mice. Our results demonstrated that forced re-expression of HNF4alpha induced the differentiation of hepatoma cells into hepatocytes, dramatically decreased "stemness" gene expression and the percentage of CD133(+) and CD90(+) cells, which are considered as cancer stem cells in HCC. Meanwhile, HNF4alpha reduced cell viability through inducing apparent apoptosis in Hep3B, while it induced cell cycle arrest and cellular senescence in HepG2. Moreover, infection of hepatoma cells by HNF4alpha abolished their tumorigenesis in mice. Most interestingly, systemic administration of adenovirus carrying the HNF4alpha gene protected mice from liver metastatic tumor formation, and intratumoral injection of HNF4alpha also displayed significant antitumor effects on transplanted tumor models. CONCLUSION: The striking suppression effect of HNF4alpha on tumorigenesis and tumor development is attained by inducing the differentiation of hepatoma cells--especially CSCs--into mature hepatocytes, suggesting that differentiation therapy with HNF4alpha may be an effective treatment for HCC patients. Our study also implies that differentiation therapy may present as one of the best strategies for cancer treatment through the induction of cell differentiation by key transcription factors.

A minimally invasive alternative for managing large pancreatic duct stones using a modified expandable metal mesh stent.

BACKGROUND AND AIM: Endoscopic clearance of large or impacted stones in the main pancreatic duct (MPD) remains a clinical challenge. In this study, we attempted to technically modify the metallic stent to facilitate the clearance of large pancreatic stones in 4 patients, hoping to lower the operative risks and shorten hospital stay. PATIENTS AND METHODS: Four patients with chronic pancreatitis and large stones in the MPD received endoscopic treatment. Inclusion criteria were: (1) pancreatic intraductal stones (number >3; diameter >or=10 mm) and strictures identified in the distal MPD; (2) calculi mainly located in the head, neck and/or body of the pancreas, and (3) failed clearance of stones using a balloon catheter or Dormia basket. Before clearing the pancreatic calculi completely, a technically modified uncovered self-expandable metallic pancreatic stent was implanted in the MPD for 4-7 days to dilate the ductal stenosis, and then drawn out through the working channel. RESULTS: As the MPD had been sufficiently expanded by the stent, the calculi were removed completely and uneventfully by the balloon or Dormia basket in all 4 patients, without inducing major postoperative complications. A 9- to 15-month follow-up did not find major complications or recurrence of large calculi in the MPD. CONCLUSIONS: Technically modified metallic stenting is a minimally invasive and clinically feasible alternative to extracorporeal shock wave lithotripsy in the management of large pancreatic duct stones.

Milder liver cirrhosis and loss of serum HBeAg do not imply lower risk for hepatocellular carcinoma development in HBV-related cirrhosis.

BACKGROUND: Serum HBeAg status and liver cirrhosis severity at the time of diagnosis of hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-related cirrhosis remain inconclusive. The aim was to investigate the status of HBeAg and cirrhosis severity at the time of HCC development in the natural history of HBV-related cirrhosis in mainland China. MATERIAL/METHODS: In a retrospective cross-sectional hospital-based setting, HBeAg status and severity of underlying cirrhosis, estimated by MELD (model for end-stage liver disease) scores and aspartate aminotransferase (AST)--to-platelet ratio index (APRI), were comprehensively compared in 377 HBsAg-positive compensated and decompensated liver cirrhosis and 434 with HCC patients to clarify the independent and joint effects of the factors. RESULTS: The majority (80.6%) of the HCC patients was negative for serum HBeAg. More than two-thirds of the patients with HCC had MELD scores <10. Severity of underlying liver cirrhosis and loss of serum HBeAg independently correlated with the risk of HCC development. Compared with the contrast group of HBeAg-positive patients with MELD scores > or =20, the odds ratio of HCC development in the patients with HBeAg negativity and MELD score <10 was 26.51 (95%CI: 8.98-78.28). CONCLUSIONS: A large proportion of HBV-related cirrhotic patients had negative serum HBeAg and mild cirrhosis severity at the time of diagnosis of HCC.

Hepatic stellate cells modulate the differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells.

Differentiation of stem cells is tightly regulated by the microenvironment which is mainly composed of nonparenchymal cells. Herein, we investigated effect of hepatic stellate cells (HSCs) in different states on mesenchymal stem cells (MSCs) differentiation. Rat HSCs were isolated and stayed quiescent within 5 days. Primary HSCs were activated by being in vitro cultured for 7 days or cocultured with Kupffer cells for 5 days. MSCs were cocultured with HSCs of different states. Expression of hepatic lineage markers was analyzed by RT-PCR and immunofluorescence. Glycogen deposition was detected by periodic acid-schiff staining. MSCs cocultured with HSC-T6 or Kupffer cell activated HSCs were morphologically transformed into hepatocyte-like cells. Hepatic-specific marker albumin was expressed in 78.3% of the differentiated MSCs 2 weeks after initiation of coculture. In addition, the differentiated MSCs also expressed alpha-fetoprotein, cytokeratin-18, glutamine synthetase and phosphoenolpyruvate carboxykinase. Glycogen deposition was detectable in 55.4% of the differentiated MSCs 6 weeks after initiation of coculture. However, the quiescent HSCs or culture activated HSCs did not exert the ability to modulate the differentiation of MSCs. Moreover, Kupffer cell activated HSCs rather than culture activated HSCs expressed hepatocyte growth factor mRNA. We draw the conclusion that fully activated HSCs could modulate MSCs differentiation into hepatocyte-like cells.

RNA interference targeting the platelet-derived growth factor receptor beta subunit ameliorates experimental hepatic fibrosis in rats.

BACKGROUND/AIMS: Platelet-derived growth factor (PDGF) is the strongest stimulator of the proliferation of hepatic stellate cells (HSCs). PDGF receptor beta subunit (PDGFR-beta) is acquired on HSCs proliferation induced by PDGF. In this study, we aim to investigate the effect of PDGFR-beta small interference RNA (siRNA) on experimental hepatic fibrosis. METHODS: We constructed a PDGFR-beta siRNA expression plasmid and investigated its effect on the activation of HSCs. Bromodeoxyuridine incorporation was performed to investigate the effect of PDGFR-beta siRNA on HSCs proliferation. A hydrodynamics-based transfection method was used to deliver PDGFR-beta siRNA to rats with hepatic fibrosis. The distribution of transgenes in the liver was observed by immunofluorescence. The antifibrogenic effect of PDGFR-beta siRNA was investigated pathologically. RESULTS: Platelet-derived growth factor receptor-beta subunit siRNA could significantly downregulate PDGFR-beta expression, suppress HSCs activation, block the mitogen-activated protein kinase signalling pathway and inhibit HSCs proliferation in vitro. PDGFR-beta siRNA expression plasmid could be delivered into activated HSCs by the hydrodynamics-based transfection method, and remarkably improve the liver function of the rat model induced by dimethylnitrosamine and bile duct ligation. Furthermore, the progression of fibrosis in the liver was significantly suppressed by PDGFR-beta siRNA in both animal models. CONCLUSIONS: Platelet-derived growth factor receptor-beta subunit siRNA may be presented as an effective antifibrogenic gene therapeutic method for hepatic fibrosis.

Inhibition of plasminogen activator inhibitor-1 expression by siRNA in rat hepatic stellate cells.

BACKGROUND AND AIM: The plasminogen activator/plasmin system is known to regulate the extracellular matrix turnover. The aim of this study was to detect the role of plasminogen activator inhibitor-1 (PAI-1) during liver fibrogenesis and investigate the functional effects of PAI-1 gene silencing in rat hepatic stellate cells (HSCs) using small interfering RNA (siRNA). METHODS: Hepatic fibrosis in rats was induced through serial subcutaneously injections of CCl(4) and the expression of PAI-1 was detected by immunohistochemistry and reverse transcription-polymerase chain reaction (PCR). PAI-1 siRNA molecules were constructed and transiently transfected into HSC-T6 using the cell suspension transfection method. The pSUPER RNA interfering system was used to establish the HSC stable cell line pSUPER-shPAI. Expression of alpha-smooth muscle actin, transforming growth factor-beta, tissue inhibitor of metalloproteinases-1, and collagen types I and III were evaluated by real-time PCR. Cell proliferation and the cell cycle were determined by the methyl thiazolyl tetrazolium (MTT) method and flow cytometry. Collagen content in HSCs supernatant was evaluated by enzyme-linked immunosorbent assay. RESULTS: The results showed that PAI-1 was upregulated during liver fibrosis, and its expression was closely correlated with the deposition of collagens. SiRNA molecules were successfully transfected into HSCs and induced inhibition of PAI-1 expression time dependently. Moreover, PAI-1 siRNA treatment downregulated alpha-smooth muscle actin, transforming growth factor-beta, tissue inhibitor of metalloproteinases-1 expression, and inhibited collagen types I and III synthesis both at the mRNA and protein level in transiently and stably transfected HSCs. CONCLUSIONS: This study suggests a significant functional role for PAI-1 in the development of liver fibrosis and that downregulating PAI-1 expression might present as a potential strategy to treat liver fibrosis.

Differentiation therapy of hepatocellular carcinoma by inhibiting the activity of AKT/GSK-3ß/ß-catenin axis and TGF-ß induced EMT with sophocarpine.

Hepatocellular carcinoma progression is thought to be driven by cancer stem cells (CSCs). No clinical trial has, as yet, shown convincing long-term disease free survival results for the majority of patients in HCC. So it is important to discover new anti-cancer agents. In our study, we chose sophocarpine, which is derived from the foxtail-like sophora herb, for its efficacy to inhibit HCC including CSCs and potential mechanism study. Our results show that sophocarpine could not only reduce HCC cell viability, eliminate HCC and reverse hepatoma cells malignant phenotype, but also reduce the ratio of CSCs and inhibit the sphere formation of CSCs in vitro. In vivo, sophocarpine significantly displayed antitumor effects in subcutaneous xenograft HCC models and orthotopic transplantation tumor models. Further studies showed that sophocarpine could exert anti-tumor effects partly via downregulating the activity of the cancer stem cell related pathways and inhibiting EMT induced by TGF-ß.